Fine-tuning function: correlation of hinge domain interactions with functional distinctions between LacI and PurR

LacI and PurR are highly homologous proteins. Their functional units are homodimers, with an N-terminal DNA binding domain that comprises the helix-turn-helix (HTH), N-linker, and hinge regions from both monomers. Hinge structural changes are known to occur upon DNA dissociation but are difficult to monitor experimentally. The initial steps of hinge unfolding were therefore examined using molecular dynamics simulations, utilizing a truncated, chimeric protein comprising the LacI HTH/N-linker and PurR hinge. A terminal Gly-Cys-Gly was added to allow "dimerization" through disulfide bond formation. Simulations indicate that differences in LacI and PurR hinge primary sequence affect the quaternary structure of the hinge x hinge' interface. However, these alternate hinge orientations would be sterically restricted by the core domain. These results prompted detailed comparison of recently available DNA-bound structures for LacI and truncated LacI(1-62) with the PurR structure. Examination revealed that different N-linker and hinge contacts to the core domain of the partner monomer (which binds effector molecule) affect the juxtapositions of the HTH, N-linker, and hinge regions in the DNA binding domain. In addition, the two full-length repressors exhibit significant differences in the interactions between the core and the C-linker connection to the DNA binding domain. Both linkers and the hinge have been implicated in the allosteric response of these repressors. Intriguingly, one functional difference between these two proteins is that they exhibit opposite allosteric response to effector. Simulations and observed structural distinctions are correlated with mutational analysis and sequence information from the LacI/GalR family to formulate a mechanism for fine-tuning individual repressor function.     

Conformational dynamics of M2 helices in KirBac channels: helix flexibility in relation to gating via molecular dynamics simulations

KirBac1.1 and 3.1 are bacterial homologues of mammalian inward rectifier K channels. We have performed extended molecular dynamics simulations (five simulations, each of >20 ns duration) of the transmembrane domain of KirBac in two membrane environments, a palmitoyl oleoyl phosphatidylcholine bilayer and an octane slab. Analysis of these simulations has focused on the conformational dynamics of the pore-lining M2 helices, which form the cytoplasmic hydrophobic gate of the channel. Principal components analysis reveals bending of M2, with a molecular hinge at the conserved glycine (Gly134 in KirBac1.1, Gly120 in KirBac3.1). More detailed analysis reveals a dimer-of-dimers type motion. The first two eigenvectors describing the motions of M2 correspond to helix kink and swivel motions. The conformational flexibility of M2 seen in these simulations correlates with differences in M2 conformation between that seen in the X-ray structures of closed channels (KcsA and KirBac) in which the helix is undistorted, and in open channels (e.g. MthK) in which the M2 helix is kinked. Thus, the simulations, albeit on a time scale substantially shorter than that required for channel gating, suggest a gating model in which the intrinsic flexibility of M2 about a molecular hinge is coupled to conformational transitions of an intracellular 'gatekeeper' domain, the latter changing conformation in response to ligand binding.     

Lactose repressor hinge domain independently binds DNA

The short 8–10 amino acid “hinge” sequence in lactose repressor (LacI), present in other LacI/GalR family members, links DNA and inducer‐binding domains. Structural studies of full‐length or truncated LacI‐operator DNA complexes demonstrate insertion of the dimeric helical “hinge” structure at the center of the operator sequence. This association bends the DNA ～40° and aligns flanking semi‐symmetric DNA sites for optimal contact by the N‐terminal helix‐turn‐helix (HtH) sequences within each dimer. In contrast, the hinge region remains unfolded when bound to nonspecific DNA sequences. To determine ability of the hinge helix alone to mediate DNA binding, we examined (i) binding of LacI variants with deletion of residues 1–50 to remove the HtH DNA binding domain or residues 1–58 to remove both HtH and hinge domains and (ii) binding of a synthetic peptide corresponding to the hinge sequence with a Val52Cys substitution that allows reversible dimer formation via a disulfide linkage. Binding affinity for DNA is orders of magnitude lower in the absence of the helix‐turn‐helix domain with its highly positive charge. LacI missing residues 1–50 binds to DNA with ～4‐fold greater affinity for operator than for nonspecific sequences with minimal impact of inducer presence; in contrast, LacI missing residues 1–58 exhibits no detectable affinity for DNA. In oxidized form, the dimeric hinge peptide alone binds to O1 and nonspecific DNA with similarly small difference in affinity; reduction to monomer diminished binding to both O1 and nonspecific targets. These results comport with recent reports regarding LacI hinge interaction with DNA sequences.     

Extrinsic interactions dominate helical propensity in coupled binding and folding of the lactose repressor protein hinge helix

A significant number of eukaryotic regulatory proteins are predicted to have disordered regions. Many of these proteins bind DNA, which may serve as a template for protein folding. Similar behavior is seen in the prokaryotic LacI/GalR family of proteins that couple hinge-helix folding with DNA binding. These hinge regions form short alpha-helices when bound to DNA but appear to be disordered in other states. An intriguing question is whether and to what degree intrinsic helix propensity contributes to the function of these proteins. In addition to its interaction with operator DNA, the LacI hinge helix interacts with the hinge helix of the homodimer partner as well as to the surface of the inducer-binding domain. To explore the hierarchy of these interactions, we made a series of substitutions in the LacI hinge helix at position 52, the only site in the helix that does not interact with DNA and/or the inducer-binding domain. The substitutions at V52 have significant effects on operator binding affinity and specificity, and several substitutions also impair functional communication with the inducer-binding domain. Results suggest that helical propensity of amino acids in the hinge region alone does not dominate function; helix-helix packing interactions appear to also contribute. Further, the data demonstrate that variation in operator sequence can overcome side chain effects on hinge-helix folding and/or hinge-hinge interactions. Thus, this system provides a direct example whereby an extrinsic interaction (DNA binding) guides internal events that influence folding and functionality.     

Structural investigation of helices II, III, and IV of B. megaterium 5S ribosomal RNA by molecular dynamics calculations.

The structures of the helices II-III region and the helix IV region of B. megaterium 5S rRNA have been examined by means of energy minimization and molecular dynamics calculations. Calculated distances between neighboring hydrogen-bonded imino protons in helices II, III, and IV were between 3.5 and 4.5 A. The overall axis for the helices II-III region is warped rather than straight. Formation of additional Watson-Crick base pairs in loop B and loop C was not evident from the atomic positions calculated by molecular dynamics. Bases in loop C are well stacked, showing no significant change during dynamics. Bulge migration in helix III does not seem to be possible; the helices II-III region prefers one conformation. Helix II is more stable than helix III. Five base pairs in helix IV were sufficiently stable to establish that helix IV is terminated by a hairpin loop of three nucleotides. U87 protrudes from loop D. Structures of the helices II-III segment and the helix IV segment of B. megaterium 5S rRNA obtained by molecular dynamics were generally consistent with the solution structure inferred from high-field proton nmr spectroscopy.     

Crystal structures of the response regulator DosR from Mycobacterium tuberculosis suggest a helix rearrangement mechanism for phosphorylation activation

The response regulator DosR is essential for promoting long-term survival of Mycobacterium tuberculosis under low oxygen conditions in a dormant state and may be responsible for latent tuberculosis in one-third of the world's population. Here, we report crystal structures of full-length unphosphorylated DosR at 2.2 Å resolution and its C-terminal DNA-binding domain at 1.7 Å resolution. The full-length DosR structure reveals several features never seen before in other response regulators. The N-terminal domain of the full-length DosR structure has an unexpected (βα)₄ topology instead of the canonical (βα)₅ fold observed in other response regulators. The linker region adopts a unique conformation that contains two helices forming a four-helix bundle with two helices from another subunit, resulting in dimer formation. The C-terminal domain in the full-length DosR structure displays a novel location of helix α10, which allows Gln199 to interact with the catalytic Asp54 residue of the N-terminal domain. In contrast, the structure of the DosR C-terminal domain alone displays a remarkable unstructured conformation for helix α10 residues, different from the well-defined helical conformations in all other known structures, indicating considerable flexibility within the C-terminal domain. Our structures suggest a mode of DosR activation by phosphorylation via a helix rearrangement mechanism.     

Intrinsic dynamics of the partly unstructured PX domain from the Sendai virus RNA polymerase cofactor P

Despite their evident importance for function, dynamics of intrinsically unstructured proteins are poorly understood. Sendai virus phosphoprotein, cofactor of the RNA polymerase, contains a partly unstructured protein domain. The phosphoprotein X domain (PX) is responsible for binding the polymerase to the nucleocapsid assembling the viral RNA. For RNA synthesis, the interplay of the dynamics of the unstructured and structured PX subdomains is thought to drive progression of the RNA polymerase along the nucleocapsid. Here we present a detailed study of the dynamics of PX using hydrogen/deuterium exchange and different NMR relaxation measurements. In the unstructured subdomain, large amplitude fast motions were found to be fine-tuned by the presence of residues with short side chains. In the structured subdomain, where fast motions of both backbone and side chains are fairly restricted, the first helix undergoes slow conformational exchange corresponding to a local unfolding event. The other two helices, which represent the nucleocapsid binding site, were found to be more stable and to reorient with respect to each other, as probed by slow conformational exchange identified for residues on the third helix. The study illustrates the intrinsically differential dynamics of this partly unstructured protein and proposes the relation between these dynamics and its function.     

Structure stability of lytic peptides during their interactions with lipid bilayers.

In this work, molecular dynamics simulations were used to examine the consequences of a variety of analogs of cecropin A on lipid bilayers. Analog sequences were constructed by replacing either the N- or C-terminal helix with the other helix in native or reverse sequence order, by making palindromic peptides based on both the N- and C-terminal helices, and by deleting the hinge region. The structure of the peptides was monitored throughout the simulation. The hinge region appeared not to assist in maintaining helical structure but help in motion flexibility. In general, the N-terminal helix of peptides was less stable than the C-terminal one during the interaction with anionic lipid bilayers. Sequences with hydrophobic helices tended to regain helical structure after an initial loss while sequences with amphipathic helices were less able to do this. The results suggests that hydrophobic design peptides have a high structural stability in an anionic membrane and are the candidates for experimental investigation.     

Molecular dynamics of individual alpha-helices of bacteriorhodopsin in dimyristol phosphatidylocholine. I. Structure and dynamics.

Understanding the role of the lipid bilayer in membrane protein structure and dynamics is needed for tertiary structure determination methods. However, the molecular details are not well understood. Molecular dynamics computer calculations can provide insight into these molecular details of protein:lipid interactions. This paper reports on 10 simulations of individual alpha-helices in explicit lipid bilayers. The 10 helices were selected from the bacteriorhodopsin structure as representative alpha-helical membrane folding components. The bilayer is constructed of dimyristoyl phosphatidylcholine molecules. The only major difference between simulations is the primary sequence of the alpha-helix. The results show dramatic differences in motional behavior between alpha-helices. For example, helix A has much smaller root-mean-squared deviations than does helix D. This can be understood in terms of the presence of aromatic residues at the interface for helix A that are not present in helix D. Additional motions are possible for the helices that contain proline side chains relative to other amino acids. The results thus provide insight into the types of motion and the average structures possible for helices within the bilayer setting and demonstrate the strength of molecular simulations in providing molecular details that are not directly visualized in experiments.     

Role of salt bridges in homeodomains investigated by structural analyses and molecular dynamics simulations

Homeodomains are a class of helix-turn-helix DNA-binding protein motifs that play an important role in the control of cellular development in eukaryotes. They fold in a three alpha-helix structural module, where the third helix is the recognition helix that fits into the major groove of DNA. Structural analysis of the members of the homeodomain family led to the identification of interactions likely to stabilize the protein domains. Linking the helices pairwise, three salt bridges were found to be well preserved within the family. Also well conserved were two cation-pi interactions between aromatic and positively charged side chains. To analyze the structural role of the salt bridges, molecular dynamics simulations (MD) were carried out on the wild-type homeodomain from the Drosophila paired protein (1fjl) and on three mutants, which lack one or two salt bridges and mimic natural mutations in other homeodomains. Analysis of the trajectories revealed only small structural rearrangements of the three helices in all MD simulations, thereby suggesting that the salt bridges have no essential stabilizing role at room temperature, but rather might be important for improving thermostability. The latter hypothesis is supported by a good correlation between the melting midpoint temperatures of several homeodomains and the number of salt bridges and cation-pi interactions that connect secondary structures.     

Side-Chain to Main-Chain Hydrogen Bonding Controls the Intrinsic Backbone Dynamics of the Amyloid Precursor Protein Transmembrane Helix

Many transmembrane helices contain serine and/or threonine residues whose side chains form intrahelical H-bonds with upstream carbonyl oxygens. Here, we investigated the impact of threonine side-chain/main-chain backbonding on the backbone dynamics of the amyloid precursor protein transmembrane helix. This helix consists of a N-terminal dimerization region and a C-terminal cleavage region, which is processed by γ-secretase to a series of products. Threonine mutations within this transmembrane helix are known to alter the cleavage pattern, which can lead to early-onset Alzheimer’s disease. Circular dichroism spectroscopy and amide exchange experiments of synthetic transmembrane domain peptides reveal that mutating threonine enhances the flexibility of this helix. Molecular dynamics simulations show that the mutations reduce intrahelical amide H-bonding and H-bond lifetimes. In addition, the removal of side-chain/main-chain backbonding distorts the helix, which alters bending and rotation at a diglycine hinge connecting the dimerization and cleavage regions. We propose that the backbone dynamics of the substrate profoundly affects the way by which the substrate is presented to the catalytic site within the enzyme. Changing this conformational flexibility may thus change the pattern of proteolytic processing.     

Side-Chain to Main-Chain Hydrogen Bonding Controls the Intrinsic Backbone Dynamics of the Amyloid Precursor Protein Transmembrane Helix

Many transmembrane helices contain serine and/or threonine residues whose side chains form intrahelical H-bonds with upstream carbonyl oxygens. Here, we investigated the impact of threonine side-chain/main-chain backbonding on the backbone dynamics of the amyloid precursor protein transmembrane helix. This helix consists of a N-terminal dimerization region and a C-terminal cleavage region, which is processed by γ-secretase to a series of products. Threonine mutations within this transmembrane helix are known to alter the cleavage pattern, which can lead to early-onset Alzheimer’s disease. Circular dichroism spectroscopy and amide exchange experiments of synthetic transmembrane domain peptides reveal that mutating threonine enhances the flexibility of this helix. Molecular dynamics simulations show that the mutations reduce intrahelical amide H-bonding and H-bond lifetimes. In addition, the removal of side-chain/main-chain backbonding distorts the helix, which alters bending and rotation at a diglycine hinge connecting the dimerization and cleavage regions. We propose that the backbone dynamics of the substrate profoundly affects the way by which the substrate is presented to the catalytic site within the enzyme. Changing this conformational flexibility may thus change the pattern of proteolytic processing.     

Self-assembly of a simple membrane protein: coarse-grained molecular dynamics simulations of the influenza M2 channel

The transmembrane (TM) domain of the M2 channel protein from influenza A is a homotetrameric bundle of α-helices and provides a model system for computational approaches to self-assembly of membrane proteins. Coarse-grained molecular dynamics (CG-MD) simulations have been used to explore partitioning into a membrane of M2 TM helices during bilayer self-assembly from lipids. CG-MD is also used to explore tetramerization of preinserted M2 TM helices. The M2 helix monomer adopts a membrane spanning orientation in a lipid (DPPC) bilayer. Multiple extended CG-MD simulations (5 × 5 μs) were used to study the tetramerization of inserted M2 helices. The resultant tetramers were evaluated in terms of the most populated conformations and the dynamics of their interconversion. This analysis reveals that the M2 tetramer has 2× rotationally symmetrical packing of the helices. The helices form a left-handed bundle, with a helix tilt angle of ∼16°. The M2 helix bundle generated by CG-MD was converted to an atomistic model. Simulations of this model reveal that the bundle's stability depends on the assumed protonation state of the H37 side chains. These simulations alongside comparison with recent x-ray (3BKD) and NMR (2RLF) structures of the M2 bundle suggest that the model yielded by CG-MD may correspond to a closed state of the channel.     

Structural rearrangements in the thyroid hormone receptor hinge domain and their putative role in the receptor function

The thyroid hormone receptor (TR) D-domain links the ligand-binding domain (LBD, EF-domain) to the DNA-binding domain (DBD, C-domain), but its structure, and even its existence as a functional unit, are controversial. The D domain is poorly conserved throughout the nuclear receptor family and was originally proposed to comprise an unfolded hinge that facilitates rotation between the LBD and the DBD. Previous TR LBD structures, however, have indicated that the true unstructured region is three to six amino acid residues long and that the D-domain N terminus folds into a short amphipathic alpha-helix (H0) contiguous with the DBD and that the C terminus of the D-domain comprises H1 and H2 of the LBD. Here, we solve structures of TR-LBDs in different crystal forms and show that the N terminus of the TRalpha D-domain can adopt two structures; it can either fold into an amphipathic helix that resembles TRbeta H0 or form an unstructured loop. H0 formation requires contacts with the AF-2 coactivator-binding groove of the neighboring TR LBD, which binds H0 sequences that resemble coactivator LXXLL motifs. Structural analysis of a liganded TR LBD with small angle X-ray scattering (SAXS) suggests that AF-2/H0 interactions mediate dimerization of this protein in solution. We propose that the TR D-domain has the potential to form functionally important extensions of the DBD and LBD or unfold to permit TRs to adapt to different DNA response elements. We also show that mutations of the D domain LXXLL-like motif indeed selectively inhibit TR interactions with an inverted palindromic response element (F2) in vitro and TR activity at this response element in cell-based transfection experiments.     

Mechanisms of domain closure in proteins

Certain enzymes respond to the binding of substrates and coenzymes by the closure of an active site that lies in a cleft between two domains. We have examined the mechanism of the domain closure in citrate synthase, for which atomic co-ordinates are available for "open" and "closed" forms. We show that the mechanism of domain closure involves small shifts and rotations of packed helices within the two domains and at their interface. Large motions of distant segments of the structure are the cumulative effect of the small relative shifts in intervening pairs of packed segments. These shifts are accommodated not by changes in packing but rather by small conformational changes in side-chains. We call this the helix interface shear mechanism of domain closure. The relative movements of packed helices follow the principles suggested by our recent study of insulin. This mechanism of domain closure is quite different from the hinge mechanisms that allow the rigid body movements of domains in immunoglobulins. The large interface between the domains of citrate synthase precludes a simple hinge mechanism for its conformational change. The helix interface shear mechanism of conformational change occurs in other enzymes that contain extensive domain-domain interfaces.     

Molecular dynamics simulations of the ErbB-2 transmembrane domain within an explicit membrane environment: comparison with vacuum simulations

Two 500-ps molecular dynamics simulations performed on the single transmembrane domain of the ErbB-2 tyrosine kinase receptor immersed in a fully solvated dilauroylphosphatidyl-ethanolamine bilayer (DLPE) are compared to vacuum simulations. One membrane simulation shows that the initial alpha helix undergoes a local pi helix conversion in the peptide part embedded in the membrane core similar to that found in simulation vacuum. Lipid/water/peptide interaction analysis shows that in the helix core, the intramolecular peptide interactions are largely dominant compared to the interactions with water and lipids whereas the helix extremities are much more sensitive to these interactions at the membrane interfaces. Our results suggest that simulations in a lipid environment are required to understand the dynamics of transmembrane helices, but can be reasonably supplemented by in vacuo simulations to explore rapidly its conformational space and to describe the internal deformation of the hydrophobic core.